Portal hypertension accounts for significant morbidity and mortality in patients with liver cirrhosis although the precise events that mediate this syndrome remain incompletely understood. The Overall Objective of this grant is to define the molecular mechanisms within the hepatic sinusoids that are responsible for portal hypertension with the goal of eventually developing new therapeutic approaches that can be tested in humans. Paracrine interactions between liver endothelial cells (LEC) and hepatic stellate cells (HSC) are required for sinusoidal vascular remodeling and contraction, which are critical steps in portal hypertension. In this regard our Preliminary Data shows that 1) LEC generate the lipid signaling molecule sphingosine-1 phosphate (S1P) which promotes HSC migration and contraction, 2) stimulation of the angiogenic growth factor receptor, fibroblast growth factor receptor 1 (FGFR) in LEC by FGF2 and FGF21 leads to S1P production, and 3) pharmacologic inhibition of LEC growth factor pathways attenuates portal hypertension development in vivo. We have parlayed these novel findings into the Central Hypothesis of the current proposal; that FGFR activation in LEC increases S1P production which stimulates HSC recruitment and contraction of hepatic sinusoids leading to portal hypertension. The Aims are to test the sub-hypotheses that: 1) LEC recruit and contract HSC through increased production and cellular release of S1P. Aim 1a will examine how both exosome and paracrine release of S1P from LEC stimulates HSC recruitment and contraction, respectively. Aim 1b will discern how FGFR signaling transcriptionally activates the S1P generating enzyme, sphingosine kinase 1 (SK1) through an epigenetic mechanism of chromatin regulation. 2) Activation of S1P production from LEC occurs through FGFR phosphorylation by FGF21 and ensuing endosome based signaling. Aim 2a will explore new mechanisms by which the endocrine FGF family member, FGF21 regulates phosphorylation of specific FGFR residues to activate FGFR signaling and S1P production. Aim 2b will focus on how FGF21 promotes FGFR internalization into intracellular endosomes to govern FGFR signaling and S1P production. 3) Inhibition of FGFR-S1P signaling in LEC attenuates development of portal hypertension in vivo by blocking recruitment and contraction of HSC. Aim 3a will dissect how the angiogenic FGFR pathway contributes to portal hypertension using a unique FGF delivery system in mice with genetic deletion of FGFR from LEC. Aim 3b will uncover the role of S1P in portal hypertension using an innovative exosome-transfer technique in mice harboring a genetic defect in S1P production. Aim 3c will test the role of FGFR internalization on portal hypertension development in vivo using a new mouse line that we recently derived. Thus this proposal will utilize a variety of innovative yet feasible, cell and molecular approaches, both in vitro and in vivo to elucidate novel pathways that contribute to portal hypertension. The results, by advancing our understanding of sinusoidal structure and function, will set a trajectory towards new and significant advances to treat portal hypertension in humans.